Chemerin Enhances Migration and Invasion of OC Cells via CMKLR1/RhoA/ROCK-Mediated EMT

Chemerin is a newly described adipokine with significant effects on obesity, metabolic disorders, and immune trafficking. Recently, chemerin has gained prominence for its potential roles in cancer and tumorigenesis with pro- or antitumor effects. To date, most referenced multifunctions of chemerin are attributed to the chemokine-like receptor 1 (CMKLR1), distributing broadly in many tissues. This study investigates the in vitro roles of chemerin treatment on migration and invasion of ovarian carcinoma cells (OVCAR-3 and SK-OV-3) and potential underlying mechanisms. Herein, exogenous chemerin treatment promotes growth and invasion of SK-OV-3 cells but has no significant effects on OVCAR-3 cells. SK-OV-3 cells undergo morphological elongation characterized by epithelial-to-mesenchymal transition (EMT) and Ras homologous genome members A (RhoA)/Rho protein-related curl spiral kinase-1 (ROCK1) activation. Furthermore, chemerin-enhanced invasion and EMT of SK-OV-3 cells are effectively blocked by C3 transferase (C3T) and Y27632 and RhoA and ROCK1 inhibitor, respectively. More importantly, RhoA/ROCK1-EMT-mediated SK-OV-3 cell invasion is orchestrated by CMKLR1 upregulation after chemerin treatment (50 ng/mL). The silencing of CMKLR1 significantly (P < 0.0001) reverses the chemerin-enhanced invasion, EMT, and RhoA/ROCK1 activation of SK-OV-3 cells. Our study indicates that chemerin promotes invasion of OC cells via CMKLR1-RhoA/ROCK1-mediated EMT, offering a novel potential target for metastasis of OC.


Introduction
As a typical gynecological malignant tumor, ovarian cancer (OC) accounts for 4% of all female tumors [1,2].Over the past decade, occurrence of OC has increased about six times, and there is increasing incidence at younger ages [3,4].Moreover, OC is especially difcult to discover during the early stage due to the lack of diagnostic sign or symptoms and efective clinical method of screening [5].Most patients will relapse within two years and the mortality rate of OC is highest among various gynecological tumors [6,7].Te poor prognosis and high rate of recurrence are associated with the aggressive metastasis of OC cells into the abdominal cavity at the later stage [8].About 85∼90% of OC are epithelial tumors, and most patients have distant peritoneal metastasis when diagnosed [9].Terefore, more research studies are urgently needed to reveal the molecular mechanisms underlying metastasis of tumor cells and explore the potential interventions to improve the survival rate of patients.
Clinical studies show that omentum, a large fat pad, is the main site for subclinical metastasis of OC and nearly 80% of patients with ovarian carcinoma appear to have omental metastasis [10].Te omentum consists of adipocytes, functioning as an endocrine organ for releasing various adipokines, and energy storage site for lipid deposition [11].It is interesting that both primary and recurrent advanced staged OC preferentially metastasize to adipose tissue, which indicates the importance of microenvironment of adipose tissue to the metastasis cascade of OC [12,13].
Chemerin, a relatively newly discovered adipokine, is mainly secreted from adipose tissue with diverse biological functions, involved in the regulation of lipid synthesis, insulin resistance, and leukocyte chemoattractant [14][15][16][17].In adipose tissue, it is found to regulate adipogenesis, lipolysis, and other metabolic activities [18].Interestingly, a number of recent studies show the role of chemerin in diferent cancers with pro-and antitumor efects depending on context [19,20].It is found that OC patients have high levels of chemerin in their ascites, which is higher than serum levels [21,22].In addition, OC tissues contain higher levels of chemerin than those in adjacent tissues [22].Some epithelial ovarian cells can synthesize and secrete chemerin, such as SK-OV-3 [23,24], indicating that chemerin might largely exert its biological efects on ovarian cells.It has been reported that chemerin promotes growth and migration of HO8910 OC cells via PD-L1 upregulation [22].An in vitro study confrms the proapoptotic role of chemerin on granulosa cells [25].However, it has also been reported that chemerin in vitro promotes or does not afect the growth of ovarian cells, such as OVCAR-3 [24,26].Tese studies indicate that although chemerin has close association with OC development, research studies on its biological efects are limited.
Chemerin plays its role mainly by activating CMKLR1 receptor that is highly expressed in OC cells [24].Chemerin induces metastasis of gastric cancer cells via CMKLR1 and CPR1/TIMPs pathways [27].But it is unknown whether chemerin alters the metastasis of OC cells through CMKLR1.Te novel fnding shows that chemerin induces the activation of transcriptional regulator serum-response factor (SRF) via the Ras homologous genome members A/Rho protein-related curl spiral kinase-1 (RhoA/ROCK1) pathway mediated by binding with CMKLR1 [28].Te RhoA/ROCK pathway is critical for metastasis of tumor cells via stimulating the phosphorylation of myosin light chain (MLC), leading to the assembly of actin [29].RhoA/ROCK activation also induces epithelial-to-mesenchymal transition (EMT) that exerts a vital role in tumor cell metastasis, especially for OC cells [30,31].However, whether chemerin regulates the metastasis of OC cells via the CMKLR1/RhoA/ ROCK1 pathway has not been investigated.Herein, we explored the potential functions of exogenous chemerin on biological activities of OC cells, which reveal a previously undocumented role of CMKLR1/RhoA/ROCK1-mediated EMT for mechanical cues in OC metastasis.

Silencing of CMKLR1.
Te siRNAs for CMKLR1 (two silencing sequences) and negative control (NC) were synthesized by GenePharma (Shanghai, China).SK-OV-3 cells were cultured into a 6-well plate and transfected with indicated CMKLR1 siRNA using Lipofectamine 8000 (Beyotime, Shanghai, China) according to manufacturer's instruction for 48 h.Ten, the silenced cells were treated with chemerin (50 ng/ml) for another 24 h.Te silencing sequences are presented in the following: si-CMKLR1#1: AAUAAUCAGGGUAUUCAUCAC si-CMKLR1#2: ACAUGUUGUGGAUGAGAAGAA 2.4.Western Blot Analysis.Cells were cultured on 6-well plates and treated with chemerin (50 ng/mL) for 24 h.After treatment, supernatants were collected and lysed with a mixture of protease inhibitor RIPA lysis bufer (Beyotime, Haimen, China) for 40 min at 4 °C.After centrifugation at 13,780 g for 10 min, the protein solution was transferred into a new tube.Te BCA kit (WLA004B, Wanleibio, China) was used to detect the protein content according to manufacturer's instruction, then it was denatured by 5x loading bufer (Solarbio, Beijing, China) at 100 °C for 5 min.Te 10-12% SDS-PAGE was prepared to separate the target proteins, and the proteins were transferred into PVDF membranes.After immersing into blocking solution for 2 h, the membranes containing targeted proteins were incubated with primary antibodies at 4 °C overnight.Ten, the membranes were treated with horseradish-peroxidase-conjugated secondary Abs after washing with PBS for three times.Te proteins were visualized after incubation with enhanced chemiluminescence (ECL) (Termo Fisher Scientifc, Rockford, IL, USA) using ChemiDoc MP chemiluminescence gel imaging system (Bio-Rad, California, USA).

RT-qPCR Analysis.
Cells were treated with chemerin (50 ng/mL) or silenced with CMKLR1 si-RNAs or RhoA/ ROCK1 inhibitors for indicated time.RNAiso plus reagent 2 International Journal of Endocrinology (Takara, Tokyo, Japan) was applied to extract the total RNA following the manufacturer's instructions.Ten, RNA (1 μg) was used for reverse transcription using PrimeScriptTMRT Master Mix (Takara).Te relative level of target mRNA was confrmed by comparing with β-actin and GAPDH mRNA according to the following conditions: initial denaturation at 95 °C for 15 min, followed by 40 cycles of 10 s denaturation at 95 °C, 5 s annealing at 60 °C, and 12 s extension at 72 °C.Te relative mRNA levels were the mean 2 −(∆∆Ct) value compared with β-actin and GAPDH.Tey were used as internal reference genes due to their highly conserved sequence and high mRNA expression in OC cells, which contributes to normalize the other gene expression.Te Primer-BLAST software (NCBI) was used to designed primers for these genes.Primer sequences, annealing temperature, and amplifcation length are shown in Table 1.
2.7.Cell Migration.Cells (10 × 10 4 /ml) were cultured on 24well dishes for 90% confuence and then were vertically scratched from top to bottom using a tip.After washing three times with PBS, cells were incubated with diferent doses of chemerin (25, 50, and 100 ng/mL) for 24 h in serumfree medium.Te cell scratches were captured at 0 and 24 h after chemerin treatment.Te cells migrated onto the scratched-of region were captured using a phase contrast microscope, and the scratched areas were quantifed using Image Pro Plus 6.0 software.Te migration ratio was calculated as follows: Migration ratio (%) � (premigratory scratched open area − postmigratory open area)/(premigratory scratched open area) × 100%.

Transwell Analysis. SK-OV-3 and OVCAR-3 cells or CMKLR1
-silenced SK-OV-3 cells were treated with chemerin (50 ng/ml) for 24 h.Ten, cells were collected and recultured (2 × 10 4 cells/200 uL) on the upper insert coated with Matrigel (BD Bioscience, san Joes, USA) of a transwell plates (Corning, NY, USA) with an 8 μm pore size flters for 4 h with the serum-free medium.Cells transferred into the lower chambers were determined and stained using 0.1% crystal violet for 30 min.Invading cells were counted using Image J software.
2.9.Statistical Analysis.At least three independent experiments were applied for data collection.All data were calculated and analyzed with GraphPad Prime software.Oneway ANOVA was applied to examine the comparison among the groups.P < 0.05 was described as the statistical signifcance.

Diferentiated Regulation of Exogenous Chemerin on Cell
Growth in OVCAR-3 and SK-OV-3 Cells.Te results of cell proliferation showed that lower dose of chemerin (25 ng/ mL) did not infuence the cell growth at any time points, but higher concentrations of chemerin (50 and 100 ng/mL) signifcantly promoted the proliferation of SK-OV-3 cells after 48 h and 72 h incubation (P < 0.0001).Furthermore, a more signifcant enhanced-growth role was observed after chemerin treatment at 50 ng/mL (Figure 1(a)).However, the diferent doses of chemerin treatment had no signifcant efect on the proliferation of OVCAR-3 cells at 24 and 48 h.Only higher concentration of chemerin (100 ng/mL) had an inhibitory role in cell growth at 72 h (Figure 1(b)).

Chemerin Enhances Migration and Invasion of SK-OV-3 Cells.
Te infuence of diferent concentrations of chemerin on cell movement in scratch and transwell processes was explored, and it was found that diferent doses of chemerin signifcantly (P < 0.0001) accelerated the healing of SK-OV-3 cells after 24 h scratch, indicating the enhanced cell migration after chemerin treatment (25, 50 and 100 ng/mL) (Figure 2(a)).Moreover, chemerin has no obvious infuence on the migration of OVCAR-3 cells at any concentration (Figure 2(b)).Consistently, the results of transwell also indicated the enhanced invasion of SK-OV-3 cells after chemerin treatment at diferent doses (Figure 2(c)).Moreover, chemerin at 50 ng/mL presented a more significant (P < 0.0001) enhancement on migration and invasion of SK-OV-3 cells (Figure 2(c)).However, chemerin did not afect invasion of OVCAR-3 cells at diferent doses (Figure 2(d)).Tese data suggest that chemerin might be inclined to regulate biological activities of the moremetastatic OC cells.

Chemerin
Activates the CMKLR1/RhoA/ROCK1 Pathway and EMT.CMKLR1 is the main binding site of chemerin in the regulation of cell behaviors; we then investigated the expression of CMKLR1 and potential downstream signaling pathway in SK-OV-3 and OVCAR-3 cells treated with chemerin (50 ng/mL) for 24 h.Te images showed that chemerin (50 ng/mL) treatment increased the cell polarity with morphological elongation of cells, the EMT characteristic, in SK-OV-3 cells (Figure 3(a)).Western blot analysis showed that chemerin addition evidently increased the expression of CMKLR1 in SK-OV-3 cells (Figures 3(b) and 3(c)).Our results showed that chemerin treatment also promoted the expression of RhoA, ROCK1, and the level of phosphorylation of MLC in SK-OV-3 cells, which attributed to cytoskeleton organization and migration/invasion, but it has no signifcant efect on protein expressions in OVCAR-3 cells (Figures 3(b) and 3(c)).Terefore, only SK-OV-3 cells were used for the subsequent experiments.Te RT-PCR analysis indicated that chemerin treatment increase the expression of Cmklr1 and the mesenchymal indicators, Ncadherin, vimentin, and Zeb1 while decreased the expression of epithelial markers, such as E-cadherin, Krt7, 19, and 14 in International Journal of Endocrinology    International Journal of Endocrinology SK-OV-3 cells (Figure 3(d)).Furthermore, chemerin treatment downregulated E-cadherin and upregulated vimentin, N-cadherin, and ZEB1 expressions in SK-OV-3 cells (Figures 3(e) and 3(f )).Tese results indicated CMKLR1-RhoA/ROCK1 and EMT might be associated with chemerinenhanced migration and invasion of SK-OV-3 cells.

Te Silencing of CMKLR1 Reverses the Chemerin-Enhanced Invasion of SK-OV-3 Cells.
To further verify that whether CMKLR1 is necessary for the efects of chemerin on RhoA/ROCK1-mediated EMT and enhanced movement of SK-OV-3 cells, the cells were transfected with CMKLR1 siRNAs before chemerin treatment.Western blot analysis showed the signifcantly (P < 0.0001) decreased CMKLR1 expression after transfection of siRNA #1 and #2 (Figures 4(a) and 4(b)).Te chemerin-enhanced expressions of RhoA, ROCK1, and the phosphorylation level of MLC were also reversed by the silencing of CMKLR1 (Figures 4(a)  and 4(b)).Consistently, the downregulation of CMKLR1 also inhibited EMT process with increased E-cadherin expression as well as decreased N-cadherin and vimentin expressions in SK-OV-3 cells treated with chemerin (Figures 4(a) and 4(c)).Moreover, the results of transwell and cell scratch also indicated that the silencing of CMKLR1 signifcantly (P < 0.0001) reduced invasion and migration of SK-OV-3 cells with less invading cells than chemerin treatment alone (Figure 4(d), 4(e), and 4(f )).Tese data proved that CMKLR1 is required for chemerin-enhanced invasion as well as the activation of RhoA/ROCK1 and EMT in SK-OV-3 cells.International Journal of Endocrinology dependent manner, cells were pretreated with either C3T (1.5 μg/ml, RhoA inhibitor), Y27632 (30 μM, ROCK1 inhibitor), or vehicle control.Te results showed that RhoA/ ROCK1 inhibition decreased the levels of MLC phosphorylation and hence verifed the action of inhibitor (Figure 5(a)).Te expression of EMT-related proteins was not activated by chemerin treatment in SK-OV-3 cells pretreated with C3T or Y27632.Chemerin decreased the expression of E-cadherin and increased expression of Ncadherin and vimentin; however, their expressions were reversed by C3T or Y27632 pretreatment (Figures 5(a) and 5(b)).Overall, these results revealed that chemerin promotes invasion of SK-OV-3 OC cells via targeting CMKLR1 that attributed to activation of RhoA/ROCK pathway-mediated EMT.

Discussion
Te intention of study was to investigate the efect of chemerin on biological behaviors of OC cells and reveal the underlying specifc mechanism.We identifed that chemerin functioned as a promotion factor for SK-OV-3 cell proliferation and migration through CMKLR1/RhoA/ROCKmediated EMT.Te present study not only enriches the researches of chemerin in female malignant tumor development especially for omental metastasis but also reveals a novel mechanism of CMKLR1/RhoA/ROCK1-mediated EMT in invasion of metastasis of OC cells.
Increasing studies show that chemerin plays a dual efect on tumor development.Chemerin expression and CMKLR1 levels have been found to be upregulated in cases of cancer cells (glioblastoma, mesothelioma, and squamous cell carcinoma of the oral tongue), exacerbating tumor expansion and metastasis [32][33][34][35].Conversely, chemerin seems to be a suppressive factor in some cases, involving acute myeloid leukemia, hepatocellular carcinoma, breast cancer, etc. [36][37][38][39][40].However, the role of chemerin in ovarian cancer cells is largely elusive.Herein, we found that chemerin has a paradoxical efect on proliferation of OVCAR-3 and SK-OV-3 cells.It was observed that chemerin (25, 50, and 100 ng/mL) treatment enhanced SK-OV-3 cell growth, but high dose of chemerin (100 ng/mL) inhibited OVCAR-3 cell growth.We infer that doses, exposure time of chemerin, or diference in type of ovarian cancer cells might account for its variation in biological function.It was reported that chemerin did not infuence the growth of ovarian noncancer cells (HOSEpiC) and OVCAR-3 and COV434 cells at doses from 3-300 ng/mL after 48 h [24].It is consistent with our results that chemerin did not afect the growth of OVCAR-3 cells after treatment for 24 and 48 h.Gao Chenxi et al. reported that chemerin addition apparently promoted cell proliferation in HO8910 OC cells but had no impact on HO8910PM cells [22].Tese data suggested that chemerin might primarily exert a distinct role in diferent types of OC cells.Furthermore, the underlying mechanism of chemerin on OC development is largely unclear.
We also found that chemerin had a biphasic role in cell movement (migration and invasion) in SK-OV-3 and OVCAR-3 cell lines.Tis may be due to the diference in migration characteristics of two cells.It has been proven that SK-OV-3 is a type of high-metastatic cell line that is more sensitive to exogenous stimuli compared to the lowmetastatic OVCAR-3 cells [41,42].A previous study has shown that the treatment of recombinant versican and hyaluronan (HA) resulted in invasion promotion in SK-OV-3 cells but had no efect on OVCAR-3 cells, which lack the HA receptor and CD44 [43].Te nitric oxide donors reduced the transmigration of SK-OV-3 cell by afecting metalloproteinase 2 (MMP2), which was not changed in OVCAR-3 cells [44].Furthermore, compared with SK-OV-  International Journal of Endocrinology 3 cells, there was no obvious morphology shift from epithelial phenotype to a more mesenchymal phenotype in OVCAR-3 cells after treatment of chemerin.Mechanistically, we found that chemerin treatment did not induce the activation of CMKLR1/RhoA/ROCK1-mediated EMT in OVCAR-3 cells, which might account for the diference in movement in two cell lines.Terefore, we speculated that the inactivation of CMKLR1/RhoA/ROCK1-mediated EMT might be the potential reason for the diference in cell movement in OVCAR-3 cells.It is unclear that how chemerin regulates biological behaviors of OC cells.Lu Zhiyuan et al. showed that chemerin addition increased proliferation, migration, and invasion of oral squamous cells carcinoma (OSCC) cells with the upregulation of antioxidative activities and EMT [45].It was demonstrated that vascular smooth muscle cell behaviors (growth and migration) were regulated by chemerin exogenous treatment via CMKLR1/lipocalin-2 axis that induced the activation of p38/MAPK and Wnt/β-catenin pathways [46].It was also reported that chemerin suppressed metastasis of hepatocellular carcinoma cells through the CMKLR1-PTEN-AKT axis [47].Our work revealed that the CMKLR1-RhoA/ROCK-EMT signaling pathway might be a novel pathway for the functions of chemerin on proliferation and invasion of OC cells.It is shown that the RhoA/ROCK1 pathway is a novel chemerin/CMKLR1 effector in mediating the activation of various transcription factors that is required for cell proliferation, migration, and invasion [48,49].Terefore, we concluded that the impact of chemerin on cell proliferation in OC cells might be related with RhoA/ROCK1 activation; however, the specifc mechanism needs to be further established.
Taken together, this work clearly illustrated the functions of chemerin on cell proliferation, migration, and invasion in OC cell.We pointed out an undocumented mechanism involving the CMKLR1/RhoA/ROCK1-mediated EMT pathway that might be a prospective indicator for the therapies of high metastasis ovarian malignant cancers.International Journal of Endocrinology

Table 1 :
Te information for primer sequences, annealing temperature, and amplifcation length.